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Abstract:

The present invention provides methods of preventing or treating
rheumatoid arthritis using a fully human antibody or antigen-binding
fragment thereof that specifically binds human interleukin-6 receptor
(hIL-6R). The methods of the present invention may include administration
of a second therapeutic agent, such as one or more of a non-steroidal
anti-inflammatory drug (NSAID), a glucocorticoid, a disease-modifying
anti-rheumatic drug (DMARD), or a TNF-alpha antagonist, T-cell blocker,
anti-CD20 antibody, an IL-1, JAK or IL-17 antagonist, or any combination
thereof.

Claims:

1. A method for treating rheumatoid arthritis (RA) in a patient, said
method comprising administering to the patient a therapeutically
effective amount of a human antibody or antigen-binding fragment thereof
which specifically binds to human interleukin-6 receptor (hIL-6R),wherein
the human antibody or antigen-binding fragment comprises the
complementarity determining regions (CDRs) of a heavy chain variable
region (HCVR) having the amino acid sequence of SEQ ID NO:19, and the
CDRs of a light chain variable region (LCVR) having the amino acid
sequence of SEQ ID NO:27.

3. The method of claim 1, wherein the antibody or antigen-binding fragment
comprises a HCVR having the amino acid sequence of SEQ ID NO:19 and a
LCVR having the amino acid sequence of SEQ ID NO:27.

4. The method of claim 1, wherein the human antibody or antigen-binding
fragment is administered to the patient subcutaneously.

5. The method of claim 1, further comprising administering a second
therapeutic agent to the patient.

6. The method of claim 5, wherein the second therapeutic agent is a
non-steroidal anti-inflammatory drug (NSAID), a glucocorticoid, a
disease-modifying anti-rheumatic drug (DMARD), a TNF-a antagonist, a
T-cell blocker, an anti-CD20 antibody, an IL-1 antagonist, or any
combination thereof.

8. The method of claim 1, wherein the therapeutically effective amount of
the human antibody or antigen-binding fragment thereof is about 50 mg to
about 200 mg.

9. The method of claim 1, wherein the patient exhibits a decrease in at
least one RA-associated biomarker at day 8 following administration of
the human antibody or antigen binding fragment as compared to the level
of the biomarker in the patient prior to the administration.

10. The method of claim 9, wherein the RA-associated biomarker is selected
from the group consisting of high-sensitivity C-reactive protein (hsCRP),
serum amyloid A (SAA), erythrocyte sedimentation rate (ESR) and serum
hepcidin.

11. The method of claim 10, wherein the RA-associated biomarker is serum
hepcidin.

12. The method of claim 11, wherein the patient exhibits at least a 60%
decrease in serum hepcidin level at day 8 following administration of the
human antibody or antigen binding fragment as compared to the level serum
hepcidin in the patient prior to the administration.

13. A method for treating rheumatoid arthritis (RA) in a patient, the
method comprising:(a) administering to the patient a first dose of a
human antibody or antigen-binding fragment thereof which specifically
binds to human interleukin-6 receptor (hIL6-R) at a first time point,
and(b) administering to the patient at least a second dose of the human
antibody or antigen-binding fragment at a second time point;wherein the
human antibody or antigen-binding fragment comprises the complementarity
determining regions (CDRs) of a heavy chain variable region (HCVR) having
the amino acid sequence of SEQ ID NO:19, and the CDRs of a light chain
variable region (LCVR) having the amino acid sequence of SEQ ID NO:27.

14. The method of claim 13, wherein the first and second doses are each
about 50 mg to about 200 mg of the human antibody or antigen-binding
fragment.

15. The method of claim 13, wherein the second time point is about 1 week
to about 6 weeks after the first time point.

17. The method of claim 13, wherein the antibody or antigen-binding
fragment comprises a HCVR having the amino acid sequence of SEQ ID NO:19
and a LCVR having the amino acid sequence of SEQ ID NO:27.

18. The method of claim 13, wherein the first and second doses of the
human antibody or antigen-binding fragment are each administered to the
patient subcutaneously.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation-in-part of U.S. patent
application Ser. No. 12/501,657, filed on Jul. 13, 2009, which is a
divisional of U.S. patent application Ser. No. 11/809,482, filed on Jun.
1, 2007, now U.S. Pat. No. 7,582,298, which claims the benefit under 35
U.S.C. §119(e) of U.S. provisional application Nos. 60/810,664,
filed on Jun. 2, 2006; and 60/843,232, filed on Sep. 8, 2006. This
application also claims the benefit under 35 U.S.C. §119(e) of U.S.
provisional application Nos. 61/181,749, filed on May 28, 2009;
61/262,661, filed on Nov. 19, 2009; and 61/297,302, filed on Jan. 22,
2010. The disclosures of all the foregoing are herein incorporated by
reference in their entireties.

FIELD OF THE INVENTION

[0002]The present invention relates to the field of therapeutic treatment
of rheumatoid arthritis. More specifically, the invention relates to the
use of interleukin-6 receptor (IL-6R) antagonists, such as anti-IL-6R
antibodies, to treat rheumatoid arthritis.

BACKGROUND

[0003]Rheumatoid arthritis (RA) is an autoimmune disease characterized by
chronic inflammation of synovial tissue, leading to destruction of the
joint architecture. It is recognized that cytokines such as tumor
necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) play
a role in joint inflammation and cartilage damage observed in RA. IL-6 is
a pleiotropic cytokine with biological effects on many cell types. IL-6
is often regarded as being downstream of TNF or IL-1 in inflammatory
cytokine cascades and may therefore represent a common pathway factor in
a wide range of inflammatory processes. Blockade of IL-6 signaling
therefore offers the potential to ameliorate multiple pathogenic features
of RA and other inflammatory diseases.

[0004]Therapeutic methods using IL-6R antagonists are mentioned in U.S.
Pat. Nos. 5,888,510; 6,723,319; and 2001/0001663. Exemplary anti-IL-6R
antibodies are described in U.S. Pat. Nos. 7,582,298; 6,410,691;
5,817,790; 5,795,695; 6,670,373; and 7,582,298.

BRIEF SUMMARY OF THE INVENTION

[0005]In a first aspect, the invention provides human antibodies,
preferably recombinant human antibodies, that specifically bind human
interleukin-6 receptor (hIL-6R). These antibodies are characterized by
binding to hIL-6R with high affinity and slow dissociation kinetics and
by the ability to neutralize IL-6 activity. The antibodies can be
full-length (for example, an IgG1 or IgG4 antibody) or may comprise only
an antigen-binding portion (for example, a Fab, F(ab')2 or scFv
fragment), and may be modified to effect functionality, e.g., to
eliminate residual effector functions (Reddy et al. (2000) J. Immunol.
164:1925-1933). In a preferred embodiment, the invention provides an
antibody or antigen-binding fragment thereof, which binds human IL-6
receptor (SEQ ID NO:1) with a KD of about 500 pM or less, as
measured by surface plasmon resonance. In a more specific embodiment, the
antibody or antigen-binding fragment has a KD of less than 300 pM,
or less than 200 pM, or even less than 100 pM. In various embodiments,
the antibody or antigen-binding fragment thereof blocks hIL-6 activity
with an IC50 of 250 pM or less, as measured by luciferase bioassay.
In more specific embodiments, the antibody or antigen-binding fragment
thereof exhibits an IC50 of 150 pM or less.

[0006]In related aspects, the antibody or antigen-binding fragment of the
invention binds hIL-6R with an affinity at least 2-fold higher than it
binds monkey IL-6R. In more preferred embodiments, the antibody or
antigen-binding fragment binds hIL-6R protein (SEQ ID NO:1) with an
affinity that is up to about 3-fold higher relative to its binding to
monkey IL-6R (Macaca fascicularis extracellular domain shown in SEQ ID
NO:251).

[0008]In a second aspect, the invention provides isolated nucleic acid
molecules that encode an antibody or antigen-binding fragment of an
antibody of the invention. In one embodiment, the nucleic acid molecule
of the invention encodes an antibody or fragment thereof comprising an
HCVR as described above. In specific embodiments, the nucleic acid
molecule encoding the HCVR is selected from the group consisting of SEQ
ID NO:2, 226, 18, 230, 34, 50, 66, 82, 98, 114, 130, 146, 238, 240, 162,
178, 234, 194 and 210, or a substantially identical sequence thereof. In
a related aspect, the invention provides an isolated nucleic acid
molecule encoding an LCVR as described above. In specific embodiments,
the nucleic acid molecule encoding the LCVR is a nucleotide sequence
selected from the group consisting of SEQ ID NO: 10, 228, 26, 232, 42,
58, 74, 90, 106, 122, 138, 154, 170, 186, 236, 202 and 218, or a
substantially identical sequence thereof.

[0016]The invention encompasses anti-hIL-6R antibodies or antigen-binding
fragments thereof having a modified glycosylation pattern. In some
applications, modification to remove undesirable glycosylation sites may
be useful, or an antibody lacking a fucose moiety on an oligosaccharide
chain, for example, to increase antibody-dependent cellular cytotoxicity
(ADCC) (see Shield et al. (2002) JBC 277:26733). In other applications,
modification of a galactosylation can be made in order to modify
complement-dependent cytotoxicity (CDC).

[0017]In further aspects, the invention provides recombinant expression
vectors carrying the nucleic acid molecules of the invention, and host
cells into which such vectors have been introduced, as are methods of
making the antibodies or antigen-binding fragments of the invention
obtained by culturing the host cells of the invention. The host cell may
be a prokaryotic or eukaryotic cell, preferably the host cell is an E.
coli cell or a mammalian cell, such as a CHO cell.

[0018]In a further aspect, the invention features a pharmaceutical
composition comprising a human antibody or antigen-binding fragment of an
antibody which specifically binds hIL-6R and a pharmaceutically
acceptable carrier.

[0019]The present invention additionally provides methods for treating
rheumatoid arthritis. The methods of the present invention comprise
administering to a patient in need of such treatment a therapeutically
effective amount of a human antibody or antigen-binding fragment of an
antibody which specifically binds to human interleukin-6 receptor
(hIL-6R).

[0020]The studies summarized in Examples 8-12 below utilize an anti-hIL-6R
antibody referred to as "mAb1." This antibody is also referred to herein
as VQ8F11-21. mAb1 (VQ8F11-21) comprises an HCVR/LCVR amino acid sequence
pair having SEQ ID NOs:19/27, and HCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3
domains represented by SEQ ID NOs:21-23-25/SEQ ID NOs:29-31-33. However,
the methods of the present invention can be practiced using any
anti-IL-6R antibody disclosed herein, as well as variants and
antigen-binding fragments of such antibody.

[0021]Examples 10-12 were designed to determine the effects of mAb1
administration on inflammation, as well as the safety and tolerability of
mAb1 in RA patients, and to determine the time course of bioeffect on
RA-associated markers after a subcutaneous dose of mAb1. As demonstrated
in Example 12, dose-dependent reduction in high-sensitivity C-reactive
protein (hsCRP) was observed through day 15 (p<0.0047). Suppression of
serum amyloid A (SAA), erythrocyte sedimentation rate (ESR) and serum
hepcidin was also observed in a dose-related manner. Significant
increases in IL-6 were also observed. At day 8, a 200 mg dose of mAb1 was
associated with median percent changes of -91.7% in hsCRP, -92.5% in SAA,
-33.8% in ESR, -66.2% in hepcidin, and +647.0% in IL-6.

[0022]Safety data from all three studies (Examples 10-12) were combined
[mAb1 (n=71) or placebo (n=24)]. During a maximum 16-week exposure
period, 16.9% and 2.4% of patients receiving mAb1 and placebo had at
least one neutrophil count of 1.0-1.5×103/uL; and 7.0% and 0%
had a neutrophil count of 0.5-1.0×103/uL. During exposure,
50.1% and 20.1% of patients receiving mAb1 and placebo had at least one
alanine aminotransferase (ALT) elevation 1-3 times the upper limit of
normal (×ULN); 1.4% and 4.2% had ALT 3-5×ULN; and 1.4% and 0%
had ALT >5×ULN. No alterations in neutrophils or liver enzymes
were associated with adverse clinical outcomes.

[0023]In summary, IL-6R inhibition with subcutaneous administration of
mAb1 was well tolerated in patients with RA with no dose-limiting
toxicities observed. Target blockade was demonstrated by the significant
increase in IL-6 after treatment. mAb1 administered to active RA patients
resulted in dose-related reduction in hsCRP, SAA, and ESR; the observed
reduction of hepcidin within one week of treatment is believed to be the
first reported demonstration of hepcidin reduction in RA in humans.
Moreover, hsCRP was suppressed for two weeks after a single 200 mg dose
of mAb1, suggesting that weekly or bi-weekly SC dosing may prove to be
efficacious.

[0025]According to certain aspects of the present invention, the
anti-hIL-6R antibody may be administered to a patient subcutaneously
(s.c.) or intravenously (iv). The anti-hIL-6R antibody may also be
administered to the patient in combination with one or more additional
therapeutic agents. In certain embodiments, the anti-hIL-6R antibody is
administered in multiple, sequential doses to a patient.

[0026]The present invention further includes the use of any of the
anti-hIL-6R antibodies, antigen-binding fragments, and/or pharmaceutical
formulations disclosed herein in the manufacture of a medicament for the
treatment, prevention and/or amelioration of rheumatoid arthritis.

[0027]Other embodiments of the present invention will become apparent from
a review of the ensuing detailed description.

DETAILED DESCRIPTION

[0028]Before the present invention is described, it is to be understood
that this invention is not limited to particular methods and experimental
conditions described, as such methods and conditions may vary. It is also
to be understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited only
by the appended claims.

[0029]Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. As used herein, the
term "about," when used in reference to a particular recited numerical
value, means that the value may vary from the recited value by no more
than 1%. For example, as used herein, the expression "about 100" includes
99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4,
etc.).

[0030]Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described. All
publications mentioned herein are incorporated herein by reference to
describe in their entirety.

Anti-hIL-6R Antibodies

[0031]The present invention includes methods that comprise administering
to a patient a human antibody, or an antigen-binding fragment thereof,
that binds specifically to hIL-6R. As used herein, the term "hIL-6R"
means a human cytokine receptor that specifically binds human
interleukin-6 (IL-6). In certain embodiments, the antibody that is
administered to the patient binds specifically to the extracellular
domain of hIL-6R. The extracellular domain of hIL-6R is shown in the
amino acid sequence of SEQ ID NO:1.

[0032]Unless specifically indicated otherwise, the term "antibody," as
used herein, shall be understood to encompass antibody molecules
comprising two immunoglobulin heavy chains and two immunoglobulin light
chains (i.e., "full antibody molecules") as well as antigen-binding
fragments thereof. The terms "antigen-binding portion" of an antibody,
"antigen-binding fragment" of an antibody, and the like, as used herein,
include any naturally occurring, enzymatically obtainable, synthetic, or
genetically engineered polypeptide or glycoprotein that specifically
binds an antigen to form a complex. Antigen-binding fragments of an
antibody may be derived, e.g., from full antibody molecules using any
suitable standard techniques such as proteolytic digestion or recombinant
genetic engineering techniques involving the manipulation and expression
of DNA encoding antibody variable and (optionally) constant domains. Such
DNA is known and/or is readily available from, e.g., commercial sources,
DNA libraries (including, e.g., phage-antibody libraries), or can be
synthesized. The DNA may be sequenced and manipulated chemically or by
using molecular biology techniques, for example, to arrange one or more
variable and/or constant domains into a suitable configuration, or to
introduce codons, create cysteine residues, modify, add or delete amino
acids, etc.

[0034]An antigen-binding fragment of an antibody will typically comprise
at least one variable domain. The variable domain may be of any size or
amino acid composition and will generally comprise at least one CDR which
is adjacent to or in frame with one or more framework sequences. In
antigen-binding fragments having a VH domain associated with a
VL domain, the VH and VL domains may be situated relative
to one another in any suitable arrangement. For example, the variable
region may be dimeric and contain VH-VH, VH-VL or
VL-VL dimers. Alternatively, the antigen-binding fragment of an
antibody may contain a monomeric VH or VL domain.

[0035]In certain embodiments, an antigen-binding fragment of an antibody
may contain at least one variable domain covalently linked to at least
one constant domain. Non-limiting, exemplary configurations of variable
and constant domains that may be found within an antigen-binding fragment
of an antibody of the present invention include: (i) VH-CH1;
(ii) VH-CH2, (iii) VH-CH3, (iv)
VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi)
VH-CH2-CH3, (vii) VH-CL; (viii)
VL-CH1; (ix) VL-CH2, (x) VL-CH3, (xi)
VL-CH1-CH2; (xii) VL-CH1-CH2-CH3;
(xiii) VL-CH2-CH3; and (xiv) VL-CL. In any
configuration of variable and constant domains, including any of the
exemplary configurations listed above, the variable and constant domains
may be either directly linked to one another or may be linked by a full
or partial hinge or linker region. A hinge region may consist of at least
2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a
flexible or semi-flexible linkage between adjacent variable and/or
constant domains in a single polypeptide molecule. Moreover, an
antigen-binding fragment of an antibody of the present invention may
comprise a homo-dimer or hetero-dimer (or other multimer) of any of the
variable and constant domain configurations listed above in non-covalent
association with one another and/or with one or more monomeric VH or
VL domain (e.g., by disulfide bond(s)).

[0036]The term "specifically binds," means that an antibody or
antigen-binding fragment thereof forms a complex with an antigen that is
relatively stable under physiologic conditions. Specific binding can be
characterized by a dissociation constant of at least about
1×10-6 M or smaller. Methods for determining whether two
molecules specifically bind are well known in the art and include, for
example, equilibrium dialysis, surface plasmon resonance, and the like.

[0037]As with full antibody molecules, antigen-binding fragments may be
monospecific or multispecific (e.g., bispecific). A multispecific
antigen-binding fragment of an antibody will typically comprise at least
two different variable domains, wherein each variable domain is capable
of specifically binding to a separate antigen or to a different epitope
on the same antigen. Any multispecific antibody format, including the
exemplary bispecific antibody formats disclosed herein, may be adapted
for use in the context of an antigen-binding fragment of an antibody of
the present invention using routine techniques available in the art.

[0038]In specific embodiments, the antibody or antibody fragment for use
in the method of the invention may be a multispecific antibody, which may
be specific for different epitopes of one target polypeptide or may
contain antigen-binding domains specific for epitopes of more than one
target polypeptide. An exemplary bi-specific antibody format that can be
used in the context of the present invention involves the use of a first
immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain,
wherein the first and second Ig CH3 domains differ from one another
by at least one amino acid, and wherein at least one amino acid
difference reduces binding of the bispecific antibody to Protein A as
compared to a bi-specific antibody lacking the amino acid difference. In
one embodiment, the first Ig CH3 domain binds Protein A and the
second Ig CH3 domain contains a mutation that reduces or abolishes
Protein A binding such as an H95R modification (by IMGT exon numbering;
H435R by EU numbering). The second CH3 may further comprise an Y96F
modification (by IMGT; Y436F by EU). Further modifications that may be
found within the second CH3 include: D16E, L18M, N44S, K52N, V57M,
and V82 I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU)
in the case of IgG1 antibodies; N44S, K52N, and V82 I (IMGT; N384S,
K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S,
K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M,
R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations
on the bi-specific antibody format described above are contemplated
within the scope of the present invention.

[0039]A "neutralizing" or "blocking" antibody, as used herein, is intended
to refer to an antibody whose binding to hIL-6R results in inhibition of
the biological activity of hIL-6. This inhibition of the biological
activity of hIL-6 can be assessed by measuring one or more indicators of
hIL-6 biological activity known to the art, such as hIL-6-induced
cellular activation and hIL-6 binding to hIL-6R (see examples below).

[0040]The fully-human anti-IL-6R antibodies disclosed herein may comprise
one or more amino acid substitutions, insertions and/or deletions in the
framework and/or CDR regions of the heavy and light chain variable
domains as compared to the corresponding germline sequences. Such
mutations can be readily ascertained by comparing the amino acid
sequences disclosed herein to germline sequences available from, for
example, public antibody sequence databases. The present invention
includes antibodies, and antigen-binding fragments thereof, which are
derived from any of the amino acid sequences disclosed herein, wherein
one or more amino acids within one or more framework and/or CDR regions
are back-mutated to the corresponding germline residue(s) or to a
conservative amino acid substitution (natural or non-natural) of the
corresponding germline residue(s) (such sequence changes are referred to
herein as "germline back-mutations"). A person of ordinary skill in the
art, starting with the heavy and light chain variable region sequences
disclosed herein, can easily produce numerous antibodies and
antigen-binding fragments which comprise one or more individual germline
back-mutations or combinations thereof. In certain embodiments, all of
the framework and/or CDR residues within the VH and/or VL
domains are mutated back to the germline sequence. In other embodiments,
only certain residues are mutated back to the germline sequence, e.g.,
only the mutated residues found within the first 8 amino acids of FR1 or
within the last 8 amino acids of FR4, or only the mutated residues found
within CDR1, CDR2 or CDR3. Furthermore, the antibodies of the present
invention may contain any combination of two or more germline
back-mutations within the framework and/or CDR regions, i.e., wherein
certain individual residues are mutated back to the germline sequence
while certain other residues that differ from the germline sequence are
maintained. Once obtained, antibodies and antigen-binding fragments that
contain one or more germline back-mutations can be easily tested for one
or more desired property such as, improved binding specificity, increased
binding affinity, improved or enhanced antagonistic or agonistic
biological properties (as the case may be), reduced immunogenicity, etc.
Antibodies and antigen-binding fragments obtained in this general manner
are encompassed within the present invention.

[0041]The term "epitope" refers to an antigenic determinant that interacts
with a specific antigen binding site in the variable region of an
antibody molecule known as a paratope. A single antigen may have more
than one epitope. Epitopes may be either conformational or linear. A
conformational epitope is produced by spatially juxtaposed amino acids
from different segments of the linear polypeptide chain. A linear epitope
is one produced by adjacent amino acid residues in a polypeptide chain.
In certain circumstance, an epitope may include moieties of saccharides,
phosphoryl groups, or sulfonyl groups on the antigen.

Therapeutic Administration and Formulations

[0042]The methods of the present invention comprise administering a
therapeutically effective amount of an anti-hIL-6R antibody to a patient.
As used herein, the phrase "therapeutically effective amount" means a
dose of anti-hIL-6R antibody that results in a detectable improvement in
one or more symptoms associated with rheumatoid arthritis or which causes
a biological effect (e.g., a decrease in the level of a particular
biomarker) that is correlated with the underlying pathologic mechanism(s)
giving rise to the condition or symptom(s) of rheumatoid arthritis. For
example, a dose of anti-hIL-6R antibody which causes an improvement in
any of the following symptoms or conditions is deemed a "therapeutically
effective amount": chronic disease anemia, fever, depression, fatigue,
rheumatoid nodules, vasculitis, neuropathy, scleritis, pericarditis,
Felty's syndrome and/or joint destruction.

[0043]In accordance with the methods of the present invention, a
therapeutically effective amount of anti-hIL-6R antibody that is
administered to the patient will vary depending upon the age and the size
(e.g., body weight or body surface area) of the patient as well as the
route of administration and other factors well known to those of ordinary
skill in the art. In certain embodiments, the dose of anti-hIL-6R
antibody administered to the patient is from about 10 mg to about 500 mg.
For example, the present invention includes methods wherein about 10 mg,
about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40
mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg,
about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95
mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg,
about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg,
about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg,
about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg,
about 200, about 205 mg, about 210 mg, about 215 mg, about 220 mg, about
225 mg, about 230 mg, about 235 mg, about 240 mg, about 245 mg, about 250
mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg,
about 280 mg, about 285 mg, about 290 mg, about 295 mg, about 300, about
325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450
mg, about 475 mg, about 500 mg, or more of anti-hIL-6R antibody is
administered to the patient.

[0044]The amount of anti-hIL-6R antibody that is administered to the
patient may be expressed in terms of milligrams of antibody per kilogram
of patient body weight (i.e., mg/kg). For example, the methods of the
present invention include administering an anti-hIL-6R antibody to a
patient at a daily dose of about 0.01 to about 100 mg/kg, about 0.1 to
about 50 mg/kg, or about 1 to about 10 mg/kg of patient body weight.

[0045]The methods of the present invention include administering multiple
doses of an anti-hIL-6R antibody to a patient over a specified time
course. For example, the anti-hIL-6R antibody can be administered about 1
to 5 times per day, about 1 to 5 times per week, about 1 to 5 times per
month or about 1 to 5 times per year. In certain embodiments, the methods
of the invention include administering a first dose of anti-hIL-6R
antibody to a patient at a first time point, followed by administering at
least a second dose of anti-hIL-6R antibody to the patient at a second
time point. The first and second doses, in certain embodiments, may
contain the same amount of anti-hIL-6R antibody. For instance, the first
and second doses may each contain about 10 mg to about 500 mg, about 20
mg to about 300 mg, about 50 mg to about 200 mg, or about 75 mg to about
150 mg of the antibody. The time between the first and second doses may
be from about a few hours to several weeks. For example, the second time
point (i.e., the time when the second dose is administered) can be from
about 1 hour to about 7 weeks after the first time point (i.e., the time
when the first dose is administered). According to certain exemplary
embodiments of the present invention, the second time point can be about
1 hour, about 4 hours, about 6 hours, about 8 hours, about 10 hours,
about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days,
about 5 days, about 6 days, about 7 days, about 2 weeks, about 4 weeks,
about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 14
weeks or longer after the first time point. Third and subsequent doses
may be similarly administered throughout the course of treatment of the
patient.

[0047]The dose may vary depending upon the age and the weight of a subject
to be administered, target disease, conditions, route of administration,
and the like. Various delivery systems are known and can be used to
administer the pharmaceutical composition of the invention, e.g.,
encapsulation in liposomes, microparticles, microcapsules, receptor
mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem.
262:4429-4432). Methods of introduction include, but are not limited to,
intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The composition may be
administered by any convenient route, for example by infusion or bolus
injection, by absorption through epithelial or mucocutaneous linings
(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together with other biologically active agents.
Administration can be systemic or local.

[0048]The pharmaceutical composition can also be delivered in a vesicle,
in particular a liposome (see Langer (1990) Science 249:1527-1533). In
certain situations, the pharmaceutical composition can be delivered in a
controlled release system, for example, with the use of a pump or
polymeric materials. In another embodiment, a controlled release system
can be placed in proximity of the composition's target, thus requiring
only a fraction of the systemic dose.

[0049]The injectable preparations may include dosage forms for
intravenous, subcutaneous, intracutaneous and intramuscular injections,
local injection, drip infusions, etc. These injectable preparations may
be prepared by methods publicly known. For example, the injectable
preparations may be prepared, e.g., by dissolving, suspending or
emulsifying the antibody or its salt described above in a sterile aqueous
medium or an oily medium conventionally used for injections. As the
aqueous medium for injections, there are, for example, physiological
saline, an isotonic solution containing glucose and other auxiliary
agents, etc., which may be used in combination with an appropriate
solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol
(e.g., propylene glycol, polyethylene glycol), a nonionic surfactant
[e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of
hydrogenated castor oil)], etc. As the oily medium, there are employed,
e.g., sesame oil, soybean oil, etc., which may be used in combination
with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
The injection thus prepared is preferably filled in an appropriate
ampoule.

[0050]Advantageously, the pharmaceutical compositions for oral or
parenteral use described above are prepared into dosage forms in a unit
dose suited to fit a dose of the active ingredients. Such dosage forms in
a unit dose include, for example, tablets, pills, capsules, injections
(ampoules), suppositories, etc. The amount of the antibody contained is
generally about 1 to 500 mg per dosage form in a unit dose; especially in
the form of injection, it is preferred that the antibody is contained in
about 5 to 100 mg and in about 10 to 250 mg for the other dosage forms.

[0051]By the phrase "therapeutically effective amount" is meant an amount
that produces the desired effect for which it is administered. The exact
amount will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques (see, for
example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical
Compounding).

[0052]In accordance with the methods of the present invention, the
anti-hIL-6R antibody (or pharmaceutical formulation comprising the
antibody) can be administered to the patient using any acceptable device
or mechanism. For example, the administration can be accomplished using a
syringe and needle or with a reusable pen and/or autoinjector delivery
device. The methods of the present invention include the use of numerous
reusable pen and/or autoinjector delivery devices to administer an
anti-hIL-6R antibody (or pharmaceutical formulation comprising the
antibody). Examples of such devices include, but are not limited to
AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen
(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX
75/25® pen, HUMALOG® pen, HUMALIN 70/30® pen (Eli Lilly and Co.,
Indianapolis, Ind.), NOVOPEN® I, II and III (Novo Nordisk, Copenhagen,
Denmark), NOVOPEN JUNIOR® (Novo Nordisk; Copenhagen, Denmark), BD®
pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN®, OPTIPEN
PRO®, OPTIPEN STARLET®, and OPTICLIK® (sanofi-aventis,
Frankfurt, Germany), to name only a few. Examples of disposable pen
and/or autoinjector delivery devices having applications in subcutaneous
delivery of a pharmaceutical composition of the present invention
include, but are not limited to the SOLOSTAR® pen (sanofi-aventis),
the FLEXPEN® (Novo Nordisk), and the KWIKPEN® (Eli Lilly), the
SURECLICK® Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET®
(Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the
HUMIRA® Pen (Abbott Labs, Abbott Park, Ill.), to name only a few.

[0053]The use of a microinfusor to deliver an anti-hIL-6R antibody (or
pharmaceutical formulation comprising the antibody) to a patient is also
contemplated herein. As used herein, the term "microinfusor" means a
subcutaneous delivery device designed to slowly administer large volumes
(e.g., up to about 2.5 mL or more) of a therapeutic formulation over a
prolonged period of time (e.g., about 10, 15, 20, 25, 30 or more
minutes). See, e.g., U.S. Pat. No. 6,629,949; U.S. Pat. No. 6,659,982;
and Meehan et al., J. Controlled Release 46:107-116 (1996). Microinfusors
are particularly useful for the delivery of large doses of therapeutic
proteins contained within high concentration (e.g., about 100, 125, 150,
175, 200 or more mg/mL) and/or viscous solutions.

Combination Therapies

[0054]The present invention includes methods of treating rheumatoid
arthritis which comprise administering to a patient in need of such
treatment an anti-hIL-6R antibody in combination with at least one
additional therapeutic agent. Examples of additional therapeutic agents
which can be administered in combination with an anti-hIL-6R antibody in
the practice of the methods of the present invention include, but are not
limited to NSAIDs, DMARDs, TNFa antagonists, T-cell blockers, CD-20
antagonists (e.g., anti-CD-20 antibodies), IL-1 antagonists, JAK
antagonists, IL-17 antagonists, and any other compound known to treat,
prevent, or ameliorate rheumatoid arthritis in a human subject. Specific,
non-limiting examples of additional therapeutic agents that may be
administered in combination with an anti-hIL-6R antibody in the context
of a method of the present invention include, but are not limited to
methotrexate, sulfasalazine, hydroxychloroquine, leflunomide, etanercept,
infliximab, adalimumab, golimumab, rilonacept, anakinra, abatacept,
certolizumab and rituximab. In the present methods, the additional
therapeutic agent(s) can be administered concurrently or sequentially
with the anti-hIL-6R antibody. For example, for concurrent
administration, a pharmaceutical formulation can be made which contains
both an anti-hIL-6R antibody and at least one additional therapeutic
agent. The amount of the additional therapeutic agent that is
administered in combination with the anti-hIL-6R antibody in the practice
of the methods of the present invention can be easily determined using
routine methods known and readily available in the art.

Biomarkers

[0055]The present invention includes methods of treating rheumatoid
arthritis by administering to a patient in need of such treatment a
therapeutically effective amount of a human antibody or antibody binding
fragment thereof which specifically binds to hIL-6R, wherein the level of
one or more RA-associated biomarkers in the patient is modified (e.g.,
increased, decreased, etc., as the case may be) following administration.
In a related aspect, the present invention includes methods for
decreasing an RA-associated biomarker in a patient by administering to
the patient a therapeutically-effective amount of a human antibody or
antigen-binding fragment thereof which specifically binds to hIL-6R.

[0056]Examples of RA-associated biomarkers include, but are not limited
to, e.g., high-sensitivity C-reactive protein (hsCRP), serum amyloid A
(SAA), erythrocyte sedimentation rate (ESR), serum hepcidin,
interleukin-6 (IL-6), and hemoglobin (Hb). As will be appreciated by a
person of ordinary skill in the art, an increase or decrease in an
RA-associated biomarker can be determined by comparing the level of the
biomarker measured in the patient at a defined time point after
administration of the anti-IL-6R antibody to the level of the biomarker
measured in the patient prior to the administration (i.e., the "baseline
measurement"). The defined time point at which the biomarker can be
measured can be, e.g., at about 4 hours, 8 hours, 12 hours, 1 day, 2
days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15
days, 20 days, 35 days, 40 days or more after administration of the
anti-hIL-6R antibody.

[0057]According to certain embodiments of the present invention, a patient
may exhibit a decrease in the level of one or more of hsCRP, SAA, ESR
and/or hepcidin following administration of an anti-hIL-6R antibody to
the patient. For example, at about day 8 following administration of a
single dose of about 200 mg of an anti-hIL-6R antibody (e.g., mAb1), the
patient may exhibit one or more of the following: (i) a decrease in hsCRP
by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
more; (ii) a decrease in SAA by about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or more; (iii) a decrease in ESR by about 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or more; and/or (iv) a decrease in
hepcidin by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or
more.

[0058]According to certain other embodiments of the present invention, a
patient may exhibit an increase in the level of one or more of Hb or IL-6
following administration of an anti-hIL-6R antibody to the patient. For
example, at about day 8 following administration of a single dose of
about 200 mg of an anti-hIL-6R antibody (e.g., mAb1), the patient may
exhibit one or more of the following: (v) an increase in Hb by about
0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0% or
more; and/or (vi) an increase in IL-6 by about 100%, 150%, 200%, 250%,
300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800% or more.

[0059]The present invention includes methods for determining whether a
subject is a suitable patient for whom administration of an anti-hIL-6R
antibody would be beneficial. For example, if an individual, prior to
receiving an anti-hIL-6R antibody, exhibits a level of an RA-associated
biomarker which signifies the disease state, the individual is therefore
identified as a suitable patient for whom administration of an
anti-hIL-6R antibody would be beneficial. According to certain exemplary
embodiments, an individual may be identified as a good candidate for
anti-hIL-6R therapy if the individual exhibits one or more of the
following: (i) a level of hsCRP greater than about 4 mg/L (e.g., about
4.5 mg/L, about 5.0 mg/L, about 5.5 mg/L, about 6.0 mg/L, about 7.0 mg/L,
about 10.0 mg/L, about 15.0 mg/L, about 20.0 mg/L, or more); (ii) a level
of SAA greater than about 3800 ng/mL (e.g., about 4000 ng/mL, 4500 ng/mL,
about 5000 ng/mL, about 5500 ng/mL, about 6000 ng/mL, about 10,000 ng/mL,
about 20,000 ng/mL, about 25,000 ng/mL, about 30,000 ng/mL, about 35,000
ng/mL, about 40,000 ng/mL, about 45,000 ng/mL, or more); (iii) an ESR
greater than about 15 mm/hr (e.g., about 16 mm/hr, about 17 mm/hr, about
18 mm/hr, about 19 mm/hr, about 20 mm/hr, about 21 mm/hr, about 22 mm/hr,
about 25 mm/hr, about 30 mm/hr, about 35 mm/hr, about 40 mm/hr, about 45
mm/hr, about 50 mm/hr, or more); and/or (iv) a level of hepcidin greater
than about 60 ng/mL (e.g., about 62 ng/mL, about 64 ng/mL, about 68
ng/mL, about 70 ng/mL, about 72 ng/mL, about 74 ng/mL, about 76 ng/mL,
about 78 ng/mL, about 80 ng/mL, about 82 ng/mL, about 84 ng/mL, about 85
ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 105 ng/mL,
or more). Additional criteria, such as other clinical indicators of RA,
may be used in combination with any of the foregoing RA-associated
biomarkers to identify an individual as a suitable candidate for
anti-hIL-6R therapy.

EXAMPLES

[0060]The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and description of
how to make and use the methods and compositions of the invention, and
are not intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperature, etc.) but some experimental
errors and deviations should be accounted for. Unless indicated
otherwise, parts are parts by weight, molecular weight is average
molecular weight, temperature is in degrees Centigrade, and pressure is
at or near atmospheric.

Example 1

Generation of Human Antibodies to Human IL-6 Receptor

[0061]Immunization of rodents can be done by any methods known in the art
(see, for example, Harlow and Lane (1988) supra; Malik and Lillehoj,
Antibody techniques: Academic Press, 1994, CA). In a preferred
embodiment, hIL-6R antigen is administered directly to mice which
comprise DNA loci encoding both human Ig heavy chain variable region and
Kappa light chain variable region (VelocImmune®, Regeneron
Pharmaceuticals, Inc.; U.S. Pat. No. 6,596,541), with an adjuvant to
stimulate the immune response. Such an adjuvant includes complete and
incomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI
(muramyl dipeptides) (see O'Hagan, Vaccine Adjuvant, by Human Press,
2000, NJ). Such an adjuvant can prevent rapid dispersal of polypeptide by
sequestering the antigen in a local depot, and may contain factors that
can stimulate host immune response. In one embodiment, hIL-6R is
administered indirectly as DNA plasmid that contains hIL-6R gene and
expresses hIL-6R using the host cellular protein expression machinery to
produce antigen polypeptide in vivo. In both approaches, the immunization
schedule requires several administrations spaced by a few weeks. The
antibody immune response is monitored by standard antigen-specific
immunoassay. When animals reached their maximum immune response, the
antibody expressing B cells were harvested and fused with mouse myeloma
cells to preserve their viability, forming hybridoma cells. To select
functionally desirable monoclonal antibodies, conditioned media of the
hybridoma cells or transfected cells were screened for specificity,
antigen-binding affinity, and potency in blocking hIL-6 binding to hIL-6R
(described below).

Example 2

Anti-hIL6R Antibodies Generated Via Direct Isolation of Splenocytes

[0062]DNA encoding VH and VL domains may be isolated directly from a
single antigen positive B cell. Briefly, the hIL-6Rα immunized
transgenic mouse was terminated and splenocytes were harvested. Red blood
cells were removed by lysis followed by pelleting the harvested
splenocytes. Resuspended splenocytes were first incubated with a cocktail
of human IgG, FITC-anti-mFc, and biotin-IL6Ra for 1 hour. The stained
cells were washed twice with PBS, then stained with a cocktail of human
and rat IgG, APC-anti-mIgM, and SA-PE for one hour. The stained cells
were washed once with PBS and were analyzed by flow cytometry on a MoFlo
(Cytomation). Each IgG positive, IgM negative, and antigen positive B
cell was sorted and plated into a separate well on a 96-well plate.
RT-PCR of antibody genes from these B cells was performed according to a
method described by Wang et al. (2000) (J Immunol Methods 244:217-225).
Briefly, cDNAs for each single B cell were synthesized via RT-PCR. Each
resulting RT product was then split and transferred into two
corresponding wells on two 96-well plates. One set of the resulting RT
products was first amplified by PCR using a 5' degenerate primer specific
for human IgG heavy chain variable region leader sequence and a 3' primer
specific for mouse heavy chain constant region, to form an amplicon. The
amplicon was then amplified again by PCR using a 5' degenerate primer set
specific for framework 1 of human IgG heavy chain variable region
sequence and a nested 3' primer specific for mouse heavy chain constant
region. The other set of the resulting RT products was first amplified by
PCR using a 5' degenerate primer specific for human kappa light chain
variable region leader sequence and a 3' primer specific for mouse kappa
light chain constant region to form an amplicon. The amplicon was then
amplified again by PCR using a 5' degenerate primer set specific for
framework 1 of human kappa light chain variable region sequence and a
nested 3' primer specific for mouse kappa light chain constant region.
The heavy chain and light chain PCR products were cloned into Sap
I-linearized antibody vectors containing IgG1 heavy chain constant region
and kappa light chain constant region, respectively. The heavy chain
plasmid has a lox2272 site and a lox511 site flanking the heavy chain
expression cassettes. In addition, immediately downstream of the lox2272
in the heavy chain plasmid there is a hygromycin-resistance gene that
lacks a promoter and an initiating ATG. The hygromycin-resistance gene is
also transcriptionally linked to a downstream eGFP gene via an IRES
sequence. The light chain plasmid has a loxP site and lox2272 site
flanking the light chain expression cassette. In addition, The light
chain plasmid has a SV40 promoter immediately before an ATG at the
lox2272 site, such that upon integration into an appropriate host cell
the lox2272-proximal SV40 promoter and initiating ATG from the light
chain plasmid is brought adjacent to the hygromycin-resistance gene in
the heavy chain plasmid in the proper reading frame to allow
transcription and translation of the hygromycin-resistance and eGFP
genes. Purified recombinant plasmids having a heavy chain variable region
sequence and plasmids having a light chain variable region sequence from
the same B cell were then combined and transfected, together with a
plasmid that expresses the Cre recombinase, into a modified CHO host cell
line. The modified CHO host cell line contains, from 5' to 3', a loxP
site, an eCFP, a lox2272 site, DsRed, and a lox511 site at a
transcriptionally active locus. Consequently, the host CHO cell can be
isolated by flow cytometry as a blue-positive, red-positive, and
green-negative cell. When recombinant plasmids expressing heavy chain and
light chain genes are transfected together with a plasmid expressing the
Cre recombinase, site-specific recombination mediated by the Cre
recombinase results in the integration of the antibody plasmids at the
chromosomal locus containing the lox sites and replacement of the eCFP
and DsRed genes. Recombinants can then be isolated as blue-negative,
red-negative, and green-positive cells by flow cytometry. Accordingly,
CHO cells transfected with recombinant plasmids having a heavy chain
variable region sequence and plasmids having a light chain variable
region sequence from the same B cell were sorted by flow cytometry, and
proper recombinants that show the blue-negative, red-negative, and
green-positive phenotype were isolated, and stable recombinant
antibody-expressing CHO cell lines were established from isolated clones.

Example 3

Antigen Binding Affinity Determination

[0063]KD of the antigen binding to the selected antibodies described
above were determined by surface kinetics on a real-time biosensor
surface plasmon resonance assay (BIAcore®). More specifically, the
affinity of the antibodies for human IL-6R was measured using a
BIAcore® 2000 or BIAcore® 3000. The antibody was captured on an
anti-mouse IgG surface and exposed to various concentrations of
recombinant hIL-6R protein either in monomeric or dimeric form. Kinetic
analysis using BIAevaluation® software was performed to obtain the
association and dissociation rate constants.

[0064]Binding affinities of the antibodies to hIL-6R was also measured for
either hybridoma-conditioned media or purified proteins by plate-based
competition immunoassay. The antibody proteins were purified using
Protein G affinity chromatography from hybridoma cell conditioning medium
that was bovine IgG-depleted (Invitrogen). For the competition ELISA,
briefly, constant amounts of antibody at different levels were premixed
with serial dilutions of antigen protein, hIL-6R-hFc, ranging from 0 to
10 μg/ml, and incubated for two hours at room temperature to reach
pseudo-binding equilibrium between the antibody and antigen. These
solutions were then transferred to 96-well hIL-6R-hFc pre-coated plates
to allow the free-antibody in the mixtures to bind to plate-coated
hIL-6R-hFc. The plates were typically coated with 1 to 2 μg/ml
hIL-6R-hFc protein in PBS solution overnight at 4° C. followed by
BSA nonspecific blocking. After washing off excess antibody in solution,
plate-bound antibodies were detected with an HRP-conjugated goat
anti-mouse IgG or IgA polyclonal antibody reagent and developed using
either colorimetric or chemiluminescence substrates. The dependency of
the signals on the concentrations of antigen in solution was analyzed
with a 4-parameter fit analysis using Prism® software (Graph Pad) and
reported as IC50. Competition immunoassay were also carried out
using steady state solution phase Kinexa® instrument (Sapidyne Inc.).

[0066]hIL-6 blocking activities of the anti-hIL-6R antibodies of the
invention were screened by hIL-6 blocking immunoassays, in vitro hIL-6
dependent cell growth bioassays, and surface plasmon resonance
(BIAcore®). The immunoassay was used to screen ability of the tested
antibody to block hIL-6 binding to hIL-6R, and the in vitro bioassay was
used to determine the potency of the antibodies in neutralizing
hIL-6R-mediated cellular signal transduction.

[0067]For the immunoassay, hIL-6 recombinant protein was coated on a
96-well plate in PBS buffer overnight at 4° C. This plate was used
to capture free hIL-6R-hFc from antibody sample solutions, and the amount
of captured hIL-6R-hFc was quantified according to the standard curve.
The sample solutions were composed of a constant amount of hIL-6R-hFc
recombinant protein (100 pM) and varying amounts of antibody, either in
crude hybridoma condition medium or as purified antibody protein, ranging
from 0 to about 50 nM in serial dilutions. The antibody-antigen mixtures
were incubated at room temperature for ˜2 hours to allow
antibody-antigen binding to reach equilibrium. The equilibrated sample
solutions were then transferred to the hIL-6 coated plates for
measurement of free hIL-6R-hFc. After 1 hour binding, the plate was
washed and bound hIL-6R-hFc was detected using HRP-conjugated goat
anti-hFc polyclonal antibodies (Jackson Immuno Research), and developed
using TMB substrate (BD Pharmigen). IC50s were determined as the
amount of antibody required to reduce 50% of IL-6R-hFc detectable to
plate bound hIL-6 ligand. Results are shown in the first column of Table
2.

[0068]Additionally, the ability of the test antibody to block hIL-6
binding to the hIL-6R receptor was determined using surface plasmon
resonance. Purified antigen hIL-6R-hFc molecules were captured by goat
anti-human IgG polyclonal antibodies immobilized on CM-5 surface through
amine coupling to a density of 250 RU. hIL-6 solution (0.25 ml, 50 nM)
was injected over the receptor surface and bound hIL-6 recorded (first
injection of IL-6). Bound hIL-6 was then removed with a pulse of 3 M
MgCl2 following by conditioning buffer. Anti-hIL6R antibody in
hybridoma conditioned medium was injected over the captured receptor
surface followed by second injection of hIL-6. The percent reduction in
hL-6 binding resulting from preformed antibody and receptor complex was
used as a score to define hIL-6 blockers from non-blockers (second
column, Table 2).

[0069]The ability of hIL-6R antibodies to block hIL-6 activity in vitro
was measured in the hIL-6-dependent myeloma line XG-1. XG-1 cells
maintained in hIL-6-containing medium were washed twice with hIL-6-free
media and cultured for ˜24 hours in hIL-6-free medium to deplete
residual hIL-6. The starved cells were then spun down and re-suspended in
the medium at 4×105 cells per ml and plated 20,000 cells per
well in a 96-well tissue culture plate. The purified antibody proteins
were serially diluted in medium and added to the plated cells at
concentrations ranging from 0 to 50 nM. Subsequently, recombinant hIL-6
was added to the wells to a final concentration of 8 pM. Cells were
allowed to grow for ˜72 hours at 37° C. in a humidified 5%
CO2 incubator. At the end of growth period, live cells were measured
using CCK-8 kit (Dojindo, Japan). IC50s were determined as described
above, and reported in the third column of Table 2.

[0070]The ability of hIL-6R antibodies to block hIL-6 activity was also
measured in vitro in the hIL-6-responsive human hepatoma cell line,
HepG2. HepG2 cells were transfected with a reporter plasmid containing a
STAT3 (Signal Transducer and activator of Transcription 3) response
element linked to a luciferase gene. The transfected cells were
trypsinized, spun down and re-suspended in the medium at approximately
2.5×105 cells per ml and plated at 20,000 cells per well in a
96-well tissue culture plate. The purified antibody proteins were
serially diluted in medium and added to the plated cells at
concentrations ranging from 0 to 100 nM. Subsequently, recombinant hIL-6
was added to the wells to a final concentration of 50 pM. The response
was measured after incubating the cells for 6 hours at 37° C. in a
humidified 5% CO2 incubator. Luciferase activity was measured with
the Steady-Glo® luciferase assay system (Promega). IC50s were
determined as described above, and reported in the fourth column of Table
2.

Example 5

Binding Epitope Diversity

[0071]An antibody binding competition immunoassay was performed using as a
control humanized antibody to human IL-6R. Briefly, a 96-well
immunosorbent plate was coated with 20 ng per well hIL-6R recombinant
protein overnight at 4° C. After blocking non-specific binding
with BSA, the hIL-6R binding sites on one half of the plate were
saturated with binding of the control antibody by addition of 500 ng of
the control per well, and to the other half of the plate was added
binding buffer only. After three hours binding at room temperature, the
purified antibodies were spiked in at a final concentration of 50 ng/ml
with and without the preexisting control antibody in the well. After one
hour of additional binding, the free antibody was washed away and the
plate-bound antibody was detected with HRP-conjugated goat anti-mouse IgG
or IgA, polyclonal antibody and the plate was developed using chromatic
HRP substrates and absorbance at 450 nm was recorded. Percentage
deductions of the binding of the anti-hIL6R antibodies by the presence of
the control antibody are listed in Table 3 below. A similar experiment
was conducted using surface plasmon resonance technology (Table 3). Both
methods generated consistent results. Antibodies VQ8F11, VV3D8, VV6A9,
VV6C10-1 bound epitopes overlapping with the control antibody; while
antibodies VQ8A9, VV1G4, VV6F12, VV7G4, VV9A6, and VV6C10-3 appeared to
bind distinct epitopes as antigen binding was not blocked by the control
antibody. Partial competition may result from steric hindrance from the
first antibody bound, even though epitopes may not be overlapping.

[0072]Four antibodies were tested for cross-reactivity to monkey IL-6R
recombinant protein using BIAcore® technology. Briefly, a biosensor
chip on which goat anti-mouse Fc polyclonal antibody was immobilized was
used to present anti-hIL-6R monoclonal antibodies to a density of about
75 RU. Recombinant human or monkey monomeric IL-6R protein (Macaca
fascicularis, extracellular domain; SEQ ID NO:251), at a concentration
range between 1.25-40 nM, was injected over the antibody surface. The
binding of the receptor to the antibody and the dissociation of the bound
complex were monitored in real-time. Both association rate constant (ka)
and dissociate rate constant (kd) were obtained, and KD calculated
(Table 4).

[0073]Among the four tested antibodies, VQ8F11, VV6A9, and VQ8A9 strongly
reacted to monkey receptor with KD values that differed by about
1.5- to about 3-fold from human receptor binding, respectively. VV1G4,
which was not blocked by the control antibody (Table 3), showed no
binding to monkey receptor despite strong binding to the human receptor
with KD of 241 pM.

Example 7

Effect of Constant Region on Binding Affinity

[0074]The binding affinity to monomeric hIL-6R of four antibodies having
mouse IgG, human IgG1 or human IgG4 (wild-type and modified) were
determined using BIAcore® as described above except a goat anti-human
Fc polyclonal antibody surface was used to capture hIgG antibodies.
Monomeric hIL-6R was injected at concentrations of 12.5, 6.25, 3.12, and
1.56 nM. The ability of the antibodies to neutralize hIL-6-dependent
HepG2/STAT3 signal transduction was also determined in a luciferase assay
(IC50). IC50s for different IgG isotypes were similar,
suggesting no effect of isotype on antibody affinity for antigen.

[0075]The pharmacokinetics of mAb1 (HCVR SEQ ID NO:19 and LCVR SEQ ID
NO:27) was examined in cynomolgus monkeys following a single subcutaneous
(SC) or IV injection at multiple dose levels. Doses for SC administration
were 1, 5, and 15 mg/kg; IV doses were 1 and 15 mg/kg. Blood samples were
collected from all animals (N=6 per group, 3 per sex per group) at
selected time points over a 52 day (1248 hr) time course. The resultant
serum samples were analyzed using a validated ELISA assay for total mAb1
concentrations. The data were analyzed by means of noncompartmental
methods. Pharmacokinetic (PK) parameter estimates such as observed
maximal concentration in serum (Cmax), the time of observed maximal
concentration (Tmax), area under the concentration vs. time curve (AUC),
clearance (CL), volume of distribution (Vz), and mean residence time
(MRT) were determined. The bioavailability following subcutaneous
administration was approximately 78%.

[0076]At low doses, mAb1 had a half-life of 28 to 30 hr. At high doses,
the terminal half-life estimate was approximately 225 hr when drug levels
were above 10 μg/ml. The half-life was approximately 80 hr when serum
concentrations were below 10 μg/ml.

Example 9

Toxicology Studies of Anti-IL-6R Antibody mAb1

[0077]In a GLP toxicology study, mAb1 was administered to cynomolgus
monkeys via SC injection twice weekly for 13 consecutive weeks (total of
26 doses). Groups of 12 animals (6/sex) were administered 0 (placebo), 1,
5, 15 or 50 mg/kg/dose. Four animals/sex scheduled for the primary
necropsy within 1 week of the end of dosing period and the remaining 2
animals/sex/group were assigned to a 12-week nondosing recovery period.
The animals were observed for mortality and moribundity. Clinical
examinations were performed daily and detailed physical examinations were
performed weekly. Individual body weights were recorded weekly. Clinical
pathology evaluations included hematology, serum chemistry, urine and CRP
analysis. Blood samples were collected periodically for toxicokinetic and
antibody evaluation. Ophthalmic and electrocardiogram examinations were
performed periodically through out the study. Complete necropsies were
performed on all animals. Selected organs were weighed and selected
tissues were examined microscopically. All animals survived to the
scheduled primary or recovery necropsies. There were no test
article-related clinical findings or effects on appetite, body weights,
opthalmologic examination results, electrocardiographic parameters,
macroscopic findings or organ weights.

[0078]Results indicated that administration of mAb1 via subcutaneous
injection to cynomolgus monkeys twice weekly for 13 consecutive weeks was
well tolerated. Slight decreases in neutrophil counts, fibrinogen, and
CRP values in monkeys administered mAb1 were considered to be effects or
possible effects of test article administration, but were not considered
to be adverse and in general, alterations in neutrophil counts and
fibrinogen values resolved during the recovery period. CRP values were
quite variable and alterations and subsequent resolution when compared to
controls were not consistent in all groups. Test article treatment
resulted in minimal to moderate perivascular mixed inflammatory cell
infiltrates in dermis and/or subcutis in subcutaneous injection sites.
Full or partial reversibility was evident following the recovery period.
Findings of unclear relationship to test article treatment included
severe diffuse subacute inflammation in heart in a single female
administered 5 mg/kg and minimal focal subacute inflammation accompanied
by mild perivascular mononuclear cell infiltrates in the brain of a
single male administered 1 mg/kg. Toxicokinetic data indicate substantial
systemic mAb1 exposure during the dosing phase of the study. In addition,
10 of the 16 recovery animals had circulating levels of mAb1 throughout
the entire recovery period, while 6 animals had no detectable mAb1 at the
end of the study. The no-observed-adverse-effect level (NOAEL) for
subcutaneous injection of mAb1 to cynomolgus monkeys twice weekly for 13
consecutive weeks was 50 mg/kg.

Example 10

Single Dose Study in Subjects with Rheumatoid Arthritis

[0079]In order to evaluate the potential of mAb1 for treatment of RA, a
study was conducted to evaluate safety, tolerability and pharmacokinetics
of SC administered mAb1. The primary objective was to assess the safety
and tolerability of a single dose of subcutaneously administered mAb1 in
subjects with rheumatoid arthritis who were concomitantly treated with
methotrexate. The secondary objective was to assess the PK profile of a
single subcutaneous dose of mAb1 and immunogenicity of a single SC dose
of mAb1.

[0080]Endpoints. The primary efficacy endpoint was the percent change from
Baseline in hs-C reactive protein ("hs-CRP"). Exploratory endpoints were
the percent change in Subject's Assessment of Pain and Subject's Global
Assessment of Disease activity.

[0081]Study Design. This is a multi-centered, randomized, double blind,
placebo-controlled, single dose parallel group study of the safety,
tolerability and pharmacodynamics of subcutaneously administered mAb1 in
subjects with rheumatoid arthritis who were receiving concomitant
methotrexate. Three (3) sequential cohorts of 5 subjects (4:1
active:placebo) were dosed SC with 50, 100, or 200 mg mAb1 or placebo. In
each cohort, 1 week safety data from the first 2 subjects dosed was
reviewed prior to dosing of the remaining subjects in the cohort.
Screening took place within the window of 2 weeks to 3 days prior to the
start of dosing (Day -14 to Day -3). On Study Day -1, the subjects
underwent pre-dose study procedures and randomization. On Study Day 1,
the subjects received SC blinded study drug or placebo. Subjects returned
home following the 8 hour blood draw. Subjects returned to the clinic for
outpatient visits on Study Days 3, 4, 8 (Week 1), 11, 15 (Week 2), 22
(Week 3), 29 (Week 4), 43 (Week 6), 57 (Week 8), 85 (Week 12) and 113
(Week 16), for safety assessments and blood sampling. Subjects completed
an End of Study (EOS) visit on Study Day 113 (Week 16). Subjects who
completed the study participated in 14 study visits.

[0082]Subject Eligibility. Inclusion Criteria: 1. Male or female=18 years
of age; 2. Subjects must weigh >50 kg and <100 kg; 3. Diagnosis of
Rheumatoid Arthritis (RA) as defined by the 1987 revised American College
of Rheumatology (ACR) criteria with disease duration of no less than 6
months and ACR class I-III; 4. Subjects must receive a minimum of 8 weeks
treatment with methotrexate (MTX) prior to the Screening visit. Subjects
must be on a stable dose of MTX (7.5 to 25 mg/week) for a minimum of 4
weeks prior to the Screening Visit; 5. All subjects will take folic acid
1 mg daily or 5 mg weekly with the MTX dose, to minimize toxicity,
according to local guidelines; 6. Oral prednisone=10 mg/day is allowed,
as long as the dose is stable for 4 weeks prior to Screening and for the
duration of the study; 7. For women of childbearing potential, a negative
serum pregnancy test at the Screening Visit (Visit 1) and a negative
urine pregnancy test at Day -1; 8. For men and women of childbearing
potential, willingness to utilize adequate contraception and not become
pregnant (or have their partner[s] become pregnant) during the full
course of the study. Adequate contraceptive measures include oral
contraceptives (stable use for 2 or more cycles prior to the Screening
visit); IUD; DEPO-PROVERA®; NORPLANT® System implants; bilateral
tubal ligation; vasectomy; condom or diaphragm plus either contraceptive
sponge, foam or jelly.

[0083]Exclusion Criteria: 1. A history of Listeriosis or active
tuberculosis (TB); 2. Persistent chronic or active recurring infection
requiring treatment with antibiotics, antivirals, or antifungals within 4
weeks prior to the Screening Visit; 3. History of prior articular or
prosthetic joint infection; 4. History of a hypersensitivity reaction,
other than localized injection site reaction (ISR), to any biological
molecule; 5. History of a hypersensitivity reaction to doxycycline,
tetracycline or related compounds; 6. Significant concomitant illness
such as, but not limited to cardiac, renal, neurological,
endocrinological, metabolic or lymphatic disease that would adversely
affect the subject's participation in this study; 7. Uncontrolled
diabetes, defined as Hemoglobin A1c (HbA1c)=9.0% at the Screening Visit;
8. Presence of any of the following laboratory abnormalities at the
Screening Visit: WBC <4,000/μl; platelet count <150,000/μl;
neutrophils <2000/μl, AST/ALT >1.5×ULN; 9. Serum
creatinine=1.5×ULN at the Screening Visit; 10. Subjects with a
positive intradermal skin tuberculin test (PPD 5TU)=5 mm induration read
at 48 to 72 hours after placement; 11. Chest radiograph (at the Screening
visit) consistent with prior tuberculosis infection including, but not
limited to, apical scarring, apical fibrosis, or multiple calcified
granulomata. This does not include non-caseating granulomata; 12. Use of
parenteral or intra-articular glucocorticoids within 4 weeks prior to the
Screening Visit; 13. Treatment with anakinra within two weeks prior to
the Screening Visit; 14. Treatment with etanercept, cyclosporine,
mycophenolate, tacrolimus, gold, penicillamine, sulfasalazine, or
hydroxychloroquine within 4 weeks prior to the Screening Visit; 15.
Treatment with adalimumab within 6 weeks prior to the Screening Visit;
16. Treatment with abatacept, azathioprine, cyclophosphamide or
infliximab within 12 weeks prior to the Screening Visit; 17. Treatment
with leflunomide or rituximab within 6 months prior to the Screening
Visit; 18. Treatment with tocilizumab or any anti-IL-6 medications prior
to Screening Visit; 19. Received administration of any live (attenuated)
vaccine within 3 months prior to the Screening Visit; 20. Known history
of Human Immunodeficiency Virus (HIV) antibody; and/or positive Hepatitis
B surface antigen (HBsAg), and/or positive Hepatitis C antibody (HCV) at
the Screening Visit; 21. History of malignancy other than carcinoma
in-situ of the cervix, or adequately treated, non-metastatic squamous or
basal cell carcinoma of the skin within five years prior to Screening
Visit; 22. History of demyelinating disease or multiple sclerosis; 23.
History of myeloproliferative disorder; 24. History of alcohol or drug
abuse within the 5 years prior to the Screening Visit; 25. Any subject
who has had surgery within 4 weeks prior to the Screening Visit; 26. Any
subjects with planned elective surgery; 27. Any other arthritic or
medical condition that in the opinion of the investigator could interfere
with study evaluations; 28. Participation in any clinical research study
evaluating another investigational drug or therapy within 30 days or at
least 5 half-lives, whichever is longer, of the investigational drug,
prior to the Screening Visit.

[0084]Study Drug Dosage and Administration. Subjects returned to the
clinic for the Baseline visit on Day -1. Once eligibility was confirmed
via pre dose procedures, the subject was randomly allocated to receive
either mAb1 or placebo. Dose assignments were determined by the
allocation schedule. Dosing took place starting at approximately 08:00 on
Day 1. Subjects were required to be fasting (no food or water) beginning
at midnight on Day -1. Study doses of 50, 100, and 200 mg were
administered via subcutaneous injection. Each dose was administered in a
single injection. All study drug injections were administered in the
abdomen.

[0085]Dose Preparation. Study drug was supplied as lyophilized powder in
sterile, single-use vials. Each vial contained 250 mg of mAb1 and
provided a stock solution of 100 mg/ml after reconstitution. Placebo was
supplied in matched vials. mAb1 was reconstituted in Sterile Water For
Injection (WFI), and contained a withdrawable volume of up to 2.0 ml.
Dosing volume was 0.5 ml for the 50 mg dose, 1.0 ml for the 100 mg dose,
and 2.0 ml for the 200 mg dose.

[0086]Study Procedures and Visits. Physical Examination. A physical
examination was conducted at the Screening visit, Day -1 (Visit 2), Day
15 (Visit 7), and Day 113 (Visit 14, EOS). Vital Signs: Vital signs
including temperature, sitting blood pressure, pulse and respiration were
collected at every study visit. On Day 1 (Visit 3), vital signs were done
prior to each pharmacokinetic blood draw at hour 0 (pre-dose) and at hour
8 post dose. Pharmacokinetic and Antibody Sample Collection Serum samples
were collected for pharmacokinetic (PK) analysis at every study visit
beginning on Day 1 (Visit 3). On Day 1 (Visit 3), samples were collected
at hour 0 (pre-dose) and at hour 8±3 minutes (post-dose). PK samples
were subsequently collected at the same time each day on Study Days 3, 4,
8, 11, 15, 22, 29, 43, 57, 85 and 113 (±2 hours). Serum samples were
collected for analysis of antibodies to mAb1.

[0087]PPD Skin Test. Tuberculin purified protein derivative (PPD) 5TU skin
test were placed intradermally at the time of the Screening visit and
read 48 to 72 hours after inoculation. All subjects, with the exception
of subjects who tested PPD positive and were successfully treated with
anti-tuberculosis therapy, but including those with a prior history of
Bacillus Calmette Guerin (BOG) administration received a PPD 5TU skin
test. Subjects' successful treatment for a prior tuberculosis infection
was documented in the source document, if applicable. Those subjects with
a positive PPD 5TU skin test, =5 mm induration at 48 to 72 hours were
excluded from the study.

[0088]Chest X-Ray. A radiologist's interpretation (signed and dated) of
the standard posterior-anterior and lateral chest X-rays noted the
absence of calcified granulomas and/or pleural scarring consistent with
TB. This information was documented in the subject's medical chart and on
the appropriate case report form at the Screening visit. A normal chest
X-ray report was deemed acceptable if it had been done within the three
months prior to the Screening visit.

[0089]Electrocardiogram. A standard 12-lead electrocardiogram (ECG) was
performed at the Screening visit, Day 3 (Visit 4), Day 8 (Visit 6), Day
15 (Visit 8) and Day 29 (Visit 10). Heart rate was recorded from the
ventricular rate and the PR, QRS, QT and QTc (QTc=QT/[60/heart rate]1/2)
intervals were recorded.

[0090]Subject's Assessment of Pain. An 11-point scale (0=no pain to
10=severe pain) was used to measure the subject's current level of pain.
The subject was instructed to circle a box on the continuum indicating
the appropriate response.

[0091]Subject's Global Assessment of Disease Activity. An 11-point scale
(0=no symptoms to 10=severe symptoms) was used to measure the subject's
overall assessment of his/her current disease activity. The subject was
instructed to circle a box on the continuum indicating the appropriate
response.

[0092]Additional Sample Collection. Blood and urine were collected as
indicated for routine laboratory measurements. Subjects fasted beginning
at midnight on the Day -1 (Visit 2, Baseline) and Day 43 (Visit 10, EOS)
study visits. Plasma and/or serum samples were collected as indicated and
used for future analysis of serum proteins (i.e. proteomic analysis) as
related to underlying disease in response to IL-6.

[0099]A second study was conducted to assess the safety and tolerability
of multiple doses of subcutaneously administered mAb1 in subjects with
rheumatoid arthritis who were receiving concomitant treatment with
methotrexate. The study was conducted in three parts and included a total
of 6 dose cohorts. Parts B and C began after the safety of Part A was
assessed.

[0102]Inclusion Criteria: 1. Male or female=18 years of age; 2. Subjects
weigh >50 kg and <100 kg; 3. Diagnosis of Rheumatoid Arthritis (RA)
as defined by the 1987 revised American College of Rheumatology (ACR)
criteria with disease duration of no less than 6 months and ACR class
I-III; 4. Subjects must have received a minimum of 12 weeks treatment
with methotrexate (MTX) prior to the Screening visit. Subjects must be on
a stable dose of MTX (7.5 to 25 mg/week) for a minimum of 6 weeks prior
to the Screening Visit; 5. All subjects took folic acid 1 mg daily or 5
mg weekly with the MTX dose, to minimize toxicity, according to local
guidelines; 6. For men and women of childbearing potential, willingness
to utilize adequate contraception and not become pregnant (or have their
partner[s] become pregnant) during the full course of the study. Adequate
contraceptive measures include oral contraceptives (stable use for 2 or
more cycles prior to the Screening visit); IUD; DEPO-PROVERA®;
NORPLANT® System implants; bilateral tubal ligation; vasectomy;
condom or diaphragm plus either contraceptive sponge, foam or jelly.

[0103]Exclusion Criteria: 1. A history of Listeriosis or active
tuberculosis (TB); 2. Persistent chronic or active recurring infection
requiring treatment with antibiotics, antivirals, or antifungals within 4
weeks prior to the Screening Visit, or any active infection at the time
of screening or randomization; 3. History of prior articular or
prosthetic joint infection; 4. History of a hypersensitivity reaction,
other than localized injection site reaction (ISR), to any biological
molecule; 5. History of a hypersensitivity reaction to doxycycline,
tetracycline or related compounds; 6. Significant concomitant illness
such as, but not limited to cardiac, renal, neurological,
endocrinological, metabolic or lymphatic disease that would adversely
affect the subject's participation in this study; 7. Uncontrolled
diabetes, defined as Hemoglobin A1c (HbA1c)=9.0% at the Screening Visit;
8. Presence of any of the following laboratory abnormalities at the
Screening Visit: WBC <4,000/L; platelet count <150,000L;
neutrophils <2000/L, AST/ALT >1.5×ULN; 9. Serum
creatinine=1.5×ULN at the Screening Visit; 10. Subjects with a
positive intradermal skin tuberculin test (PPD 5TU)=5 mm induration read
at 48 to 72 hours after placement; 11. Chest radiograph (at the Screening
visit) consistent with prior tuberculosis infection including, but not
limited to, apical scarring, apical fibrosis, or multiple calcified
granulomata. This does not include non-caseating granulomata; 12. Use of
oral prednisone or equivalent >10 mg per day within 4 weeks prior to
the Screening Visit; 13. Use of parenteral or intra-articular
glucocorticoids within 4 weeks prior to the Screening Visit; 14.
Treatment with anakinra within two weeks prior to the Screening Visit;
15. Treatment with etanercept, cyclosporine, mycophenolate, tacrolimus,
gold, penicillamine, sulfasalazine, or hydroxychloroquine within 4 weeks
prior to the Screening Visit; 16. Treatment with adalimumab within 6
weeks prior to the Screening Visit; 17. Treatment with abatacept,
azathioprine, cyclophosphamide or infliximab within 12 weeks prior to the
Screening Visit; 18. Treatment with leflunomide or rituximab within 6
months prior to the Screening Visit; 19. Use of tocilizumab or any other
anti-IL-6 medication prior to the Screening Visit; 20. Prior exposure to
mAb1; 21. Received administration of any live (attenuated) vaccine within
3 months prior to the Screening Visit; 22. Known history of Human
Immunodeficiency Virus (HIV) antibody; and/or positive Hepatitis B
surface antigen (HBsAg), and/or positive Hepatitis C antibody (HCV) at
the Screening Visit; 23. History of malignancy other than adequately
treated carcinoma in-situ of the cervix, or adequately treated,
non-metastatic squamous or basal cell carcinoma of the skin within five
years prior to Screening Visit; 24. History of demyelinating disease or
multiple sclerosis; 25. History of myeloproliferative disorder; 26.
History of alcohol or drug abuse within the 5 years prior to the
Screening Visit; 27. Any subject who has had surgery within 4 weeks prior
to the Screening Visit; 28. Any subjects with planned elective surgery;
29. Any other arthritic or medical condition that in the opinion of the
investigator could interfere with study evaluations; 30. Participation in
any clinical research study evaluating another investigational drug or
therapy within 30 days or at least 5 half-lives, whichever is longer, of
the investigational drug, prior to the Screening Visit.

[0104]Study drug was supplied as a lyophilized powder in sterile,
single-use vials. Each vial contained 269 mg of mAb1 and provided a stock
solution of 100 mg/mL after reconstitution. Placebo was supplied in
matched vials. mAb1 was reconstituted in Sterile Water for Injection
(WFI), and contained a withdrawable volume of up to 2 mL.

[0105]Study Drug Dosage. Subjects had a Screening Visit on Day -14 to Day
-3. Once eligibility was confirmed, the subjects were randomly allocated
to receive either mAb1 or placebo. Dose assignment into each group was
determined by an IVRS. In Part A, subjects were randomized into two dose
cohorts. Subjects in dose cohort 1 received either 50 mg mAb1 SC every
week or matching placebo every week and subjects in cohort 2 received
either 100 mg mAb1 SC alternating with placebo every week or matching
placebo every week. Subjects in dose cohort 2 received a dose of 100 mg
SC mAb1 on Day 1 (Visit 2), Day 15 (Visit 4) and Day 29 (Visit 6) and a
dose of placebo on Day 8 (Visit 3) and Day 22 (Visit 5). Escalation to
Part B took place after all 20 subjects in Part A completed the Day 36
(Week 5) visit and the laboratory and safety data were reviewed, and the
safety of the 200 mg single dose was confirmed. Subjects in Part B were
randomized into three dose cohorts. Subjects in dose cohort 3 received
either 100 mg mAb1 SC every week or matching placebo every week; subjects
in cohort 4 received either 200 mg mAb1 SC alternating with placebo every
week or matching placebo every week and subjects in dose cohort 5
received either 150 mg mAb1 SC every week or matching placebo every week.
Subjects in dose cohort 4 received a dose of 200 mg SC mAb1 on Day 1
(Visit 2), Day 15 (Visit 4) and Day 29 (Visit 6) and a dose of placebo on
Day 8 (Visit 3) and Day 22 (Visit 5).

[0109]A third study was conducted to assess the bioeffect of a single dose
of mAb1 compared with placebo in subjects with active rheumatoid
arthritis who were receiving concomitant treatment with methotrexate.

[0110]Study Design. The study was designed as a single-dose, double-blind,
placebo-controlled, parallel group safety, tolerability and
pharmacodynamic study of subcutaneously (SC) administered mAb1 in
rheumatoid arthritis patients who are receiving concomitant methotrexate.
Four (4) parallel groups of 8 subjects each with active rheumatoid
arthritis were dosed SC with 50, 100 or 200 mg mAb1 or placebo (1:1:1:1).
Each subject received a single dose of mAb1 or placebo, and was followed
for 6 weeks. Subjects (32) completed 10 study visits: (Screening, Day 1,
Day 4, Day 8, Day 12, Day 15, Day 22, Day 29, Day 36 and Day 43).

[0111]Inclusion Criteria: 1. Male or female=18 years of age; 2. Subjects
must weigh >50 kg and <100 kg; 3. Diagnosis of Rheumatoid Arthritis
(RA) as defined by the 1987 revised American College of Rheumatology
(ACR) criteria with disease duration of no less than 6 months and ACR
class I-III; 4. Subjects must receive a minimum of 12 weeks treatment
with methotrexate (MTX) prior to the Screening visit. Subjects must be on
a stable dose of MTX (7.5 to 25 mg/week) for a minimum of 8 weeks prior
to the Screening Visit; 5. All subjects will take folic acid at 5 mg
weekly or greater with the MTX dose, to minimize toxicity; 6. hs-CRP=10
mg/L; 7. For men and women of childbearing potential, willingness to
utilize adequate contraception and not become pregnant (or have their
partner[s] become pregnant) during the full course of the study. Adequate
contraceptive measures include oral contraceptives (stable use for 2 or
more cycles prior to screening) and other prescription pharmaceutical
contraceptives; IUD; bilateral tubal ligation; vasectomy; condom or
diaphragm plus either contraceptive sponge, foam or jelly.

[0112]Exclusion Criteria: 1. A history of Listeriosis or active
tuberculosis (TB); 2. Persistent chronic or active recurring infection
requiring treatment with antibiotics, antivirals, or antifungals within 4
weeks prior to the Screening Visit; 3. History of prior articular or
prosthetic joint infection; 4. History of a hypersensitivity reaction,
other than localized injection site reaction (ISR), to any biological
molecule; 5. History of a hypersensitivity reaction to doxycycline,
tetracycline or related compounds; 6. Significant concomitant illness
such as, but not limited to cardiac, renal, neurological,
endocrinological, metabolic or lymphatic disease that would adversely
affect the subject's participation in this study; 7. Uncontrolled
diabetes, defined as Hemoglobin A1c (HbA1c)=9.0% at the Screening Visit;
8. Presence of any of the following laboratory abnormalities at the
Screening Visit: WBC <4,000/μ; platelet <150,000/μl;
neutrophils <2000/μl, AST/ALT >1.5×ULN; 9. Serum
creatinine=1.5×ULN at the Screening Visit; 10. Subjects with a
positive intradermal skin tuberculin test=5 mm induration read at 48 to
72 hours after placement; 11. Chest radiograph (at the Screening visit)
consistent with prior tuberculosis infection including, but not limited
to, apical scarring, apical fibrosis, or multiple calcified granulomata.
This does not include non-caseating granulomata; 12. Treatment with oral
prednisone or equivalent >10 mg per day within 4 weeks prior to the
Screening Visit; 13. Use of parenteral or intra-articular glucocorticoids
within 4 weeks prior to the Screening Visit; 14. Treatment with anakinra
within two weeks prior to the Screening Visit; 15. Treatment with
etanercept, cyclosporine, mycophenolate, tacrolimus, gold, penicillamine,
sulfasalazine, or hydroxychloroquine within 4 weeks prior to the
Screening Visit; 16. Treatment with adalimumab within 6 weeks prior to
the Screening Visit; 17. Treatment with abatacept, azathioprine,
cyclophosphamide or infliximab within 12 weeks prior to the Screening
Visit; 18. Treatment with leflunomide or rituximab within 6 months prior
to the Screening Visit; 19. Treatment with tocilizumab or any other
anti-IL-6 medication prior to Screening Visit; 20. Start treatment or
change dose of current treatment with NSAIDs/COX2 inhibitors for 2 weeks
prior to Screening; 21. Received administration of any live (attenuated)
vaccine within 3 months prior to the Screening Visit; 22. Known history
of Human Immunodeficiency Virus (HIV) antibody; and/or positive Hepatitis
B surface antigen (HBsAg), and/or positive Hepatitis C antibody (HCV) at
the Screening Visit; 23. History of malignancy other than carcinoma
in-situ of the cervix, or adequately treated, non-metastatic squamous or
basal cell carcinoma of the skin within five years prior the Screening
Visit; 24. History of alcohol or drug abuse within the 5 years prior to
the Screening Visit; 25. Any subject who has had surgery within 4 weeks
prior to the Screening Visit; 26. Any subjects with planned elective
surgery; 27. Participation in any clinical research study evaluating
another investigational drug or therapy within 30 days or at least 5
half-lives, whichever is longer, of the investigational drug, prior to
the Screening Visit; 28. Previous exposure to mAb1.

[0113]Study Drug Dosage. Subjects had a Screening Visit on Day -14 to Day
-3. Once eligibility was confirmed, the subjects were randomly allocated
to receive either mAb1 or placebo. Subjects were enrolled in 4 parallel
groups of 8 subjects each and were dosed with 50, 100 or 200 mg SC mAb1
or placebo (1:1:1:1). Each subject received a single SC dose of mAb1 or
placebo on Day 1, and was followed for 6 weeks.

[0114]Dose Preparation. Study drug was supplied as lyophilized powder in
sterile, single-use vials. Each vial contained 269 mg of mAb1 and
provided a stock solution of 100 mg/ml after reconstitution. The maximum
dose to be administered per vial was 200 mg. Placebo was supplied in
matched vials. mAb1 was reconstituted in Sterile Water For Injection
(WFI), and contained a withdrawable volume of up to 2 ml. The 50 mg dose
was administered at 0.5 ml, 100 mg at 1 ml, 200 mg at 2 ml and the
placebo at 2 ml. All study drug injections were administered in the
abdomen.

[0122]The present invention is not to be limited in scope by the specific
embodiments describe herein. Indeed, various modifications of the
invention in addition to those described herein will become apparent to
those skilled in the art from the foregoing description. Such
modifications are intended to fall within the scope of the appended
claims.